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Cathepsin L2 Levels Inversely Correlate with Skin Color

Journal of Investigative Dermatology (2006) 126, 2345–2347. doi:10.1038/sj.jid.5700409; published online 25 May 2006

TO THE EDITOR lightly (LK) or darkly (DK) pigmented that keratinocytes derived from lightly While skin color is the most notable skins) (Cascade Biologics, Portland, pigmented skins (LK) have about difference among ethnic skins, many OR). When evaluating expression levels 3.5-fold increase in CTSL2 protein other differences exist in the structure of (chip results, Table S1), levels (normalized by b-actin) relative and function of ethnic skins. Unfortu- only cathepsin L2 (CTSL2) was found to to those of darkly pigmented skins (DK) nately, the current knowledge of phy- be differentially expressed in relation to siological and pathological properties pigmentary skin backgrounds, and was (Figure 2a). In addition, CTSL2 protein of the skin is based mainly on Cauca- about 7.5-folds higher in keratinocytes levels in human skins with different sian skin studies (reviewed in Taylor, from lightly pigmented, relative to levels of pigment deposition (as shown 2002). Elucidating differences among darkly pigmented skins (Table S1). by Fontana–Mason staining, Figure 2b and c) were analyzed by immunohisto- ethnic skins is not only of great biolo- CTSL2 is a lysosomal cysteine chemistry staining. In agreement gical importance, but also of great (Adachi et al., 1998; Santamaria et al., with the CTSL2 mRNA levels in ethnic interest in skin care improvement. 1998; Bromme et al., 1999). It is skins, CTSL2 protein levels were Keratinocytes play an important role expressed in primary keratinocytes, found to be higher in lighter skins, and in skin physiology, affecting skin barrier melanocytes, and HaCaT keratinocytes strongly reduced in darker skins function, immune responses and (Figure 1a), but not in primary fibro- (Figure 2d and e). wound-healing processes (Williams blasts (Figure 1b). The differential ex- To date, there are no specific in- and Kupper, 1996). Earlier studies pression of CTSL2 mRNA in documented the role of the keratinocyte hibitors to CTSL2; therefore, we could keratinocytes (but not of other cathe- not directly measure CTSL2 activity in receptor PAR-2 in the regulation of skin psins and of cystatins, the natural keratinocyte’s derived from skins of pigmentation (Seiberg et al., 2000a, b; inhibitors of cathepsins) was confirmed different colors. The total enzymatic Paine et al., 2001), and the differential by reverse transcriptase (RT)-PCR activity of cathepsins was measured expression of the PAR-2 pathway in (Figure 1c; Table S2 in Supplementary using substrate Z-Phe-Arg-AMC keratinocytes of different ethnic origins material for primers). Interestingly, (Bachem, Torrance, CA). Substrate (Babiarz-Magee et al., 2004). In order melanocytes from different ethnic hydrolysis catalyzed by cathepsins to identify other differences in kerati- origins expressed the same levels of was monitored by fluorescent emission nocytes’ expression profiles, we per- CTSL2 mRNA (Figure 1d). (excitation at 360 nm and emission at formed DNA chip analysis of Western blot analysis of keratino- 465 nm). Cell lysate of keratinocytes keratinocytes obtained from different cytes using polyclonal antibodies from lightly pigmented skins showed skin types (four individuals of either specific to CTSL2 (Figure S1) showed 1.570.16-fold higher cathepsin activity than keratinocytes derived from darker Abbreviations: CTSL2, cathepsin L2; RT, reverse transcriptase skins (data were pooled from three

www.jidonline.org 2345 N Chen et al. Cathepsin L2 Levels Inversely Correlate with Skin Color

a b a LK DK

GAPDHCTSL2CTSL CTSB MKH CTSL2 CTSL2 GAPDH -Actin

cdLK DK bc CTSL2

CTSL LM DM CTSL2 CTSB Cystatin A CTSL CTSB Cystatin B GAPDH de Cystatin M

GAPDH

Figure 1. CTSL2 mRNA expression is higher in keratinocytes from lighter-colored skins. (a) RNA was isolated from primary neonatal keratinocytes (K), melanocytes (M), and from HaCaT cells (H). CTSL2 expression was analyzed by RT-PCR. Figure 2. CTSL2 protein levels inversely correlate with skin color. (a) Keratinocytes obtained from lightly (b) RNA was isolated from primary fibroblasts pigmented skin (LK) have higher levels of CTSL2 protein than those of darkly pigmented skins (DK). The and analyzed by RT-PCR for CTSL2, CTSL, and CTSL2 polyclonal antibodies recognize three bands by Western blot analysis, corresponding to the three CTSB expression. (c) Neonatal keratinocytes from processed forms of CTSL2 protein in cells. Protein lysates (15 mg) of dark or light keratinocytes were used lightly and darkly pigmented skins (LK and DK, to detect CTSL2, and 2 mg of the same lysates were used to detect b-actin. The experiment was carried out respectively) were analyzed by RT-PCR for twice with four different lots of keratinocytes. Representative data are shown. (b–e) Human skin biopsies expression levels of CTSL, CTSB, CTSL2, were obtained from the Cooperative Human Tissue Network Eastern Division (Philadelphia, PA). Skin and cystatins A, B, and C (the endogenous pigment deposition levels were documented by Fontana–Mason staining. (b and c) Examples of lightly physiological inhibitors of cathepsins). Only and darkly pigmented skins are presented. (d and e) Immunohistochemistry was performed with CTSL2 was differentially expressed in anti-CTSL2 antibodies using skin sections of same individuals (b and d, c and e, are each from the keratinocytes of different skin colors. The same individual). Sections were counterstained with hematoxylin (Vector Labs, Burlingame, CA). experiment was repeated three times with CTSL2 protein levels are higher in (d) lighter skins, and are strongly reduced in (e) darker skins. different lots of keratinocytes. Representative data Immunohistochemistry was repeated twice, with total of 16 skin sections, eight each from lightly and are shown. (d) Neonatal melanocytes from lightly darkly pigmented skins. Representative pictures are shown. Bar ¼ 0.05 mm. and darkly pigmented skins (LM and DM, respectively) were analyzed by RT-PCR for CTSL2 enzymatic activity is higher in plasia with hyperproliferation of basal expression levels of CTSL, CTSB, and CTSL2. No light-skin-derived keratinocytes. epidermal keratinocytes, acanthosis, differential expression was identified in melanocytes of different skin colors. The Cathepsins are a large family of and hyperkeratosis, but no appreciable experiment was repeated three times with lysosomal cysteine , which changes in hair pigmentation (Roth different lots of melanocytes. Representative data are involved in numerous cellular et al., 2000; Benavides et al., 2002; are shown. Primers used in this figure are listed in processes. CTSL2 shares 80% protein Tobin et al., 2002). This defective skin Table S2. sequence identity with CTSL, but its and hair phenotype was largely rescued expression is mostly confined to the by human keratin 14 promoter driven thymus, testis, and cornea (Adachi CTSL2 (Hagemann et al., 2004). These independent experiments). CA074, a et al., 1998; Santamaria et al., 1998; data suggest that the CTSL2 proteolytic specific inhibitor, reduced Bromme et al., 1999). In skin, CTSL2 activities play a role in the maintenance keratinocytes cathepsin B activity by was isolated from human stratum cor- of homeostasis of both the epidermis B70%. Cathepsin activities of CA074- neum (Watkinson, 1999; Bernard et al., and the hair follicles. While the differ- treated keratinocytes were about 2.2- 2003) and was shown to have a high ential expression of CTSL2 was discov- fold higher in light-skin-derived kerati- caseinolytic activity, which is distinct ered in relation to skin color, we could nocytes versus dark ones (for Materials from that of CTSL. To date, no CTSL2 not find evidence for the differential and Methods, see Supplementary mate- ortholog has been identified in mice, expression of CTSL2 in melanocytes, or rials). Since overall cathepsin expression and the human CTSL2 protein is more for the regulation of melanogenesis via (excluding CSTL2) is similar in both homologous to the mouse CTSL than to CTSL2. Our data demonstrate ethnic dark and light keratinocytes (Table S1 the human CTSL sequence (Bromme skin differences that are not directly and Figure 1), the difference of the et al., 1999). ctslÀ/À mice exhibit a associated with melanogenesis, ex- remaining cathepsin activity represents complex skin phenotype consisting of panding on ethnic skin differences CTSL2. This suggests that the total periodic hair loss and epidermal hyper- beyond the pigmentary process.

2346 Journal of Investigative Dermatology (2006), Volume 126 N Chen et al. Cathepsin L2 Levels Inversely Correlate with Skin Color

Ashy skin is a condition that affects Development (PRD), and Laura Babiarz-Magee of Hagemann S, Gunther T, Dennemarker J, Loh- Johnson & Johnson CPPW, for DNA chip analysis. muller T, Bromme D, Schule R et al. (2004) many dark-skinned individuals. Ashi- Dr N. Fusenig (Heidelberg, Germany) provided The human ness is described as a common physio- HaCaT Cells. Special thanks to Dr Robin can compensate for murine in logical skin condition, induced by Thurmond from The Johnson & Johnson PRD for mouse epidermis and hair follicles. Eur J Cell environmental influence, and in parti- providing purified recombinant CTSL and CTSL2 Biol 83:775–80 proteins. Tissue samples were provided by the cular by cold and dry weather. In dry Paine C, Sharlow E, Liebel F, Eisinger M, Shapiro Cooperative Human Tissue Network, which is S, Seiberg M (2001) An alternative approach conditions, light reflectance from dead funded by the National Cancer Institute. to depigmentation by soybean extracts via stratum corneum cells of dark skins Nannan Chen1, Miri Seiberg1 and inhibition of the PAR-2 pathway. J Invest results in a dull, ashy look. Ashy skin Connie B. Lin1 Dermatol 116:587–95 has seldom been studied, but is known Roth W, Deussing J, Botchkarev VA, Pauly-Evers 1 to be unrelated to inflammation or to The Johnson & Johnson Skin Research Center, M, Saftig P, Hafner A et al. (2000) Cathepsin CPPW, a division of Johnson & Johnson L deficiency as molecular defect of furless: skin pathology (Uhoda et al., 2003). Consumer Companies Inc., Skillman, New hyperproliferation of keratinocytes and per- The development of ashiness might Jersey, USA. E-mail: [email protected] tubation of hair follicle cycling. FASEB J result from defects in corneodesmolysis 14:2075–86 Santamaria I, Velasco G, Cazorla M, Fueyo A, and desquamation (Sato et al., 1998), in SUPPLEMENTARY MATERIAL which enzymatic activities of proteases, Campo E, Lopez-Otin C (1998) Cathepsin L2, Figure S1. The CTSL2 polyclonal antibodies are a novel human cysteine proteinase produced particularly serine- and cathepsin-like specific to CTSL2. by breast and colorectal carcinomas. Cancer , might be altered. Here, we Table S1. Arbitrary expression value of cathepsins Res 58:1624–30 show that CTSL2 is differentially ex- selected from microarray analysis of keratinocytes Sato J, Denda M, Nakanishi J, Koyama J (1998) pressed in ethnic skins, with lower derived from light and dark skins. Dry condition affects desquamation of stra- levels of mRNA, protein, and enzy- Table S2. PCR primers used in Figure 1. tum corneum in vivo. J Dermatol Sci 18:163–9 matic activity in darkly pigmented Materials and Methods Seiberg M, Paine C, Sharlow E, Andrade-Gordon skins. While the physiological function P, Costanzo M, Eisinger M et al. (2000a) of CTSL2 in human skin remains to be REFERENCES Inhibition of melanosome transfer results explored, we hypothesize that reduced Adachi W, Kawamoto S, Ohno I, Nishida K, in skin lightening. J Invest Dermatol proteolytic activity within the stratum Kinoshita S, Matsubara K et al. (1998) Isola- 115:162–7 tion and characterization of human cathepsin corneum of darker skins is involved, at Seiberg M, Paine C, Sharlow E, Andrade-Gordon V: a major proteinase in corneal epithelium. P, Costanzo M, Eisinger M et al. (2000b) The least in part, in the creation of the ‘‘ashy Invest Ophthalmol Vis Sci 39:1789–96 protease-activated receptor 2 regulates pig- skin phenotype’’. CTSL2 has been Babiarz-Magee L, Chen N, Seiberg M, Lin CB mentation via keratinocyte-melanocyte inter- identified as a human stratum corneum (2004) The expression and activation of actions. Exp Cell Res 254:25–32 desquamation processing-related en- protease-activated receptor-2 correlate with Taylor SC (2002) Skin of color: biology, structure, skin color. Pigment Cell Res 17:241–51 zyme (Bernard et al., 2003), further function, and implications for dermatologic Benavides F, Starost MF, Flores M, Gimenez-Conti disease. J Am Acad Dermatol 46:S41–62 supporting our hypothesis. This work IB, Guenet JL, Conti CJ (2002) Impaired hair Tobin DJ, Foitzik K, Reinheckel T, Mecklenburg L, highlights the importance of under- follicle morphogenesis and cycling with Botchkarev VA, Peters C et al. (2002) The standing ethnic skin at a deeper level abnormal epidermal differentiation in nackt lysosomal protease cathepsin L is an impor- than skin color. Johnson & Johnson mice, a cathepsin L-deficient mutation. Am J tant regulator of keratinocyte and melanocyte Pathol 161:693–703 differentiation during hair follicle morpho- CPPW, a division of Johnson & Johnson Bernard D, Mehul B, Thomas-Collignon A, Simo- genesis and cycling. Am J Pathol 160: Consumer Companies Inc. approved all netti L, Remy V, Bernard MA et al. (2003) 1807–21 described studies. Analysis of proteins with caseinolytic activity Uhoda E, Pierard-Franchimont C, Petit L, Pierard in a human stratum corneum extract revealed G (2003) Skin weathering and ashiness in a yet unidentified cysteine protease and black Africans. Eur J Dermatol 13:574–8 CONFLICT OF INTEREST identified the so-called ‘‘stratum corneum This research was supported and funded by thiol protease’’ as cathepsin l2. J Invest Watkinson A (1999) Stratum corneum thiol Johnson & Johnson CPPW, a division of Johnson Dermatol 120:592–600 protease (SCTP): a novel cysteine protease & Johnson Consumer Companies Inc. The authors of late epidermal differentiation. Arch Der- are employed by the company. Bromme D, Li Z, Barnes M, Mehler E (1999) matol Res 291:260–8 Human cathepsin V functional expression, tissue distribution, electrostatic surface po- Williams IR, Kupper TS (1996) Immunity at ACKNOWLEDGMENTS tential, enzymatic characterization, and the surface: homeostatic mechanisms of the skin immune system. Life Sci 58: We thank Drs Andrew Carmen and Xuejun Liu of chromosomal localization. Biochemistry 1485–507 Johnson & Johnson Pharmaceutical Research and 38:2377–85

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